Quantitative Analysis of Ascorbic Acid and Isoascorbic Acid in Foods by High-Performance Liquid Chromatography with Electrochemical Detection

Abstract

A procedure is presented for the direct and simultaneous determination of ascorbic acid (AA) and isoascorbic acid (IAA) in food products by paired-ion reverse-phase high-performance liquid chromatography. Three Supelcosil C18 columns were used with a pH 5.4 mobile phase containing 0.04 M sodium acetate, 5 mM tetrabutylammonium hydrogen sulfate and 0.015% metaphosphoric acid. Food samples, preserved with metaphosphoric acid and diluted with mobil phase, were injected (20μI) using an autosampler. Detection of AA and IAA was by amperometry using a glassy carbon electrode and Ag/AgCl reference electrode. The applied potential was +0.6 volt and the sensitivity was 20 nA. As little as 0.5 ng of each component could be detected under these conditions. When the same samples were incubated with homocysteine to reduce dehydroascorbic acid (DHAA) and dehydroisoascorbic acid (DHIAA) to AA and IAA respectively and reinjected into the system the values for total AA and IAA were obtained. The concentration of the oxidized forms of DHAA and DHIAA could then be calculated by substraction.